The linker histone H1–BRCA1 axis is a crucial mediator of replication fork stability

The replication-dependent histones H1 interact with BRCA1 upon replication stress. Cells deficient for H1 fail to recruit BRCA1 to stalled replication forks and undergo fork resection and collapse.

Full guidelines are available on our Instructions for Authors page, https://www.life-science-alliance.org/authors We encourage our authors to provide original source data, particularly uncropped/-processed electrophoretic blots and spreadsheets for the main figures of the manuscript. If you would like to add source data, we would welcome one PDF/Excel-file per figure for this information. These files will be linked online as supplementary "Source Data" files. ***IMPORTANT: It is Life Science Alliance policy that if requested, original data images must be made available. Failure to provide original images upon request will result in unavoidable delays in publication. Please ensure that you have access to all original microscopy and blot data images before submitting your revision.*** ---------------------------------------------------------------------------Reviewer #1 (Comments to the Authors (Required)): In this manuscript, the authors show that histone H1 variants interact with BRCA1 in a replication-stress-dependent manner and Transient loss of the replication-dependent histones H1 leads to the accumulation of stalled replication intermediates. This is linked to BRCA1 recruitment failure leading to Mre11-dependent degradation of nascent DNA. This is an interesting work presenting original results. Additional experiments are required to consolidate the data presented.
Major critiques: -It is unclear whether BRCA1-H1 interaction takes place on chromatin. This could be addressed by proximity ligation assay (PLA).
-The authors should check whether RAD51 chromatin binding is affected by the downregulation of histone H1 -The authors should verify whether the downregulation of H1 affects nascent DNA stability in one of the BRCA1-defective cancer cell models. This is an interesting manuscript that identifies an interaction between histone H1 and BRCA1 and points to a role for H1 in fork protection. More in details, the authors found that BRCA1 interacts with the linker histone H1 variants upon treatment with HU. Upon knock-down of H1, BRCA1 fails to be recruited to chromatin despite normal activation of the DDR. The authors then elegantly use DNA fiber assay to study the dynamics of DNA replication forks upon siRNA of Histone H1 in the presence and absence of HU and demonstrate that loss of H1 leads to fork resection, dependent on MRE11. Overall, I think the experiments are well conducted and the findings will be of interest to the field. Thus, I am overall favorable for publication of this study. Having said that, some aspects could be improved.
Authors could investigate the pathways that support this interaction to strengthen their findings. Work from the Mailand lab showed that histone H1 ubiquitination is involved in DSB repair via RNF8 (Thorslund et al., Nature, 2015). How interaction between H1 and BRCA1 is modulated by this pathway ? and what about the intra-S-phase checkpoint ? (is interaction abrogated by treatment with ATRi ? or is it prevented by knock-down of RNF8 ?) It's interesting that H1 knock-down does not change fork speed. I was wondering if reduction of histone H1 also affected the number of origins given the reduced chromatin compaction ? Accordingly to authors model, siRNA of histone H1 should lead to a "BRCAness". This aspect is very exciting. Have authors tested sensitivity to PARP inhibitors ?
Reviewer #3 (Comments to the Authors (Required)): In the present manuscript Ozgencil et al. report the identification of a novel and replication stress dependent interaction between BRCA1 and the linker histone H1 and propose a role for histone H1 in replication fork stability. The interaction was identified in a previous mass spec from the same authors and proposed to be dependent on CK2 phosphorylation. However, mutation of BRCA1 on this specific residue does not abrogate this interaction, suggesting the presence of multiple interaction sites.
Consistent with a functional role for this interaction silencing of histone H1 by siRNA prevented BRCA1 localization to sites of DNA damage while not preventing yH2AX phosphorylation. Given the previously established link between BRCA1 and fork protection they show that loss of H1 induces fork degradation, all dependent on the MRE11 nuclease.
The work is interesting given the increased attention to histone H1 and its roles in regulating chromatin structure. The manuscript is well written and data are clearly presented. I have a few minor comments that authors should address to fully support publication.
It would be good to further strengthen the finding that this interaction and function does not depend on a specific histone H1 subtype. I understand that this issue is complicated by the different expression levels (in different cell lines) of the H1 variants but authors could strengthen this by: 1) analyse the interaction between BRCA1 and another histone H1 subtype, different from H1.3, at the endogenous levels (and/or at least in a different cell line ?).
2) The authors showed that expression of siRNA resistant GFP-H1.3 rescue BRCA1 recruitment. Could they see a similar effect with another histone H1 gene overexpression? this would strengthen their finding that this effect is not H1.3-specific.
3) IP with flag 1.4 seems weaker. Is this a wb exposure issue? a plasmid expression problem or might depend on different properties of H1.4 ? 4) are there other potential CK2 sites involved which might explain authors results? is this interaction reduced by CK2 inhibition for example? (this should be at least discussed) 1 st Authors' Response to Reviewers June 5, 2023 1 Reviewer #1 (Comments to the Authors (Required)): In this manuscript, the authors show that histone H1 variants interact with BRCA1 in a replication-stress-dependent manner and Transient loss of the replication-dependent histones H1 leads to the accumulation of stalled replication intermediates. This is linked to BRCA1 recruitment failure leading to Mre11-dependent degradation of nascent DNA. This is an interesting work presenting original results. Additional experiments are required to consolidate the data presented.
First of all, we would like to thank the reviewer for their positive comments on our work and the experimental suggestions. We believe we have addressed their comments in full: Major critiques: -It is unclear whether BRCA1-H1 interaction takes place on chromatin. This could be addressed by proximity ligation assay (PLA).

To address the reviewer comment we have now performed PLA assays using antibodies against endogenous BRCA1 and H1.2 (see revised Fig. 1F) and H1.3 (see revised Fig. S1D). Our new data show that interaction between endogenous BRCA1 and H1 takes place on chromatin and is specifically induced by HU treatment.
-The authors should check whether RAD51 chromatin binding is affected by the downregulation of histone H1 To address the reviewer comment we have performed chromatin fractionation (upon silencing of histone H1 and treatment with HU) and analysed Rad51 levels (see revised Fig. S2B). Our new data show that loss of histone H1 impairs Rad51 chromatin loading upon treatment with HU. This is in agreement with the role of BRCA1 in promoting Rad51-dependent stabilization of stalled replication forks.
-The authors should verify whether the downregulation of H1 affects nascent DNA stability in one of the BRCA1-defective cancer cell models.
To address the reviewer comment we have now performed DNA fiber/fork protection assays in UWB1.289 ovarian cancer cells harbouring BRCA1 mutations. Importantly, we have also performed the same experiments in UWB1.289 complemented with WT BRCA1. As shown in our revised manuscript (see Fig. 4C) treatment with HU leads to a reduction of the IdU/CldU ratio in UWB1.289 cells, in agreement with the established role of BRCA1 in fork protection. However, this effect is not worsened by silencing of histone H1, pointing to an epistatic mechanism. In agreement with this, UWB1.289 complemented with wt BRCA1 showed restored fork protection, an effect that was lost upon silencing of histone H1. To further corroborate the genetic and mechanistic interaction between H1 and BRCA1 we have also performed co-silencing experiments in U2OS. As shown in revised Fig. S4, silencing of H1 and BRCA1 leads to a similar decrease in the IdU/CldU ratio (fork deprotection). Importantly this effect was not worsened by H1 and BRCA1 co-depletion, pointing to an epistatic mechanism.
Minor critiques -The colony survival assay in Fig 5B should be better explained. Is the downregulation of H1 shown here stable over several cell cycles?
We apologize with the reviewer for the lack of clarity. Colony forming assays were done upon transient silencing of histone H1 (which is stable for at least 72 hours in our experiments). We clarified this in the revised text.
Reviewer #2 (Comments to the Authors (Required)): We would like to sincerely thank the reviewer for their enthusiastic support. We have addressed their minor comments below.
This is an interesting manuscript that identifies an interaction between histone H1 and BRCA1 and points to a role for H1 in fork protection. More in details, the authors found that BRCA1 interacts with the linker histone H1 variants upon treatment with HU. Upon knockdown of H1, BRCA1 fails to be recruited to chromatin despite normal activation of the DDR. The authors then elegantly use DNA fiber assay to study the dynamics of DNA replication forks upon siRNA of Histone H1 in the presence and absence of HU and demonstrate that loss of H1 leads to fork resection, dependent on MRE11. Overall, I think the experiments are well conducted and the findings will be of interest to the field. Thus, I am overall favorable for publication of this study. Having said that, some aspects could be improved.
Authors could investigate the pathways that support this interaction to strengthen their findings. Work from the Mailand lab showed that histone H1 ubiquitination is involved in DSB repair via RNF8 (Thorslund et al., Nature, 2015). How interaction between H1 and BRCA1 is modulated by this pathway ? and what about the intra-S-phase checkpoint ? (is interaction abrogated by treatment with ATRi ? or is it prevented by knock-down of RNF8 ?) To strengthen our manuscript and address the reviewer comments we have now further investigated the mechanistic basis of the H1-BRCA1 interaction. We now show that interaction between H1 and BRCA1 takes place specifically on chromatin (Revised Fig.  1F and S1D) upon HU treatment and requires both the C-terminal domain (containing the CK2 phosphorylation site S1336) and the ring domain of BRCA1 (Revised Fig. S1A). These data suggest that interaction between H1 and BRCA1 involves multiple sites. In agreement with this and to address the reviewer comment we now also show that transient silencing of RNF8 strongly reduces interaction between endogenous H1 and BRCA1 (revised Fig. S1E). This data points to a critical role for H1 ubiquitination in regulating binding to and recruitment of BRCA1 to stressed replication forks. Treatment with ATR inhibitors is known to reduce fork speed and induce fork stalling while affecting hundreds of substrates within the DDR. We believe it would be very difficult to raise any conclusions based on the use of such inhibitors. We hope the reviewer will agree with our conclusions.
It's interesting that H1 knock-down does not change fork speed. I was wondering if reduction of histone H1 also affected the number of origins given the reduced chromatin compaction ?
We thank the reviewer for the experimental suggestion. We have now analysed inter origin distance values upon silencing of H1 by DNA fiber assay and found that downregulation of the replication-dependent histone H1 variants does not affect origin activation (as shown by absence of IOD changes, see revised Fig. 3C) Accordingly to authors model, siRNA of histone H1 should lead to a "BRCAness". This aspect is very exciting. Have authors tested sensitivity to PARP inhibitors ?
We thank the reviewer for the comment and experimental suggestion. Indeed, we now show that transient silencing of histone H1 leads to sensitization to PARP inhibitors, in agreement with our "H1-BRCA1 axis" model (see revised Fig. 5C).

Reviewer #3 (Comments to the Authors (Required)):
We would like to thank the reviewer for their supporting comments and constructive feedback. We have addressed all their minor comments as follows: In the present manuscript Ozgencil et al. report the identification of a novel and replication stress dependent interaction between BRCA1 and the linker histone H1 and propose a role for histone H1 in replication fork stability. The interaction was identified in a previous mass spec from the same authors and proposed to be dependent on CK2 phosphorylation. However, mutation of BRCA1 on this specific residue does not abrogate this interaction, suggesting the presence of multiple interaction sites. Consistent with a functional role for this interaction silencing of histone H1 by siRNA prevented BRCA1 localization to sites of DNA damage while not preventing yH2AX phosphorylation. Given the previously established link between BRCA1 and fork protection they show that loss of H1 induces fork degradation, all dependent on the MRE11 nuclease.
The work is interesting given the increased attention to histone H1 and its roles in regulating chromatin structure. The manuscript is well written and data are clearly presented. I have a few minor comments that authors should address to fully support publication.
It would be good to further strengthen the finding that this interaction and function does not depend on a specific histone H1 subtype. I understand that this issue is complicated by the different expression levels (in different cell lines) of the H1 variants but authors could strengthen this by: 1) analyse the interaction between BRCA1 and another histone H1 subtype, different from H1.3, at the endogenous levels (and/or at least in a different cell line ?).
We thank the reviewer for the experimental suggestion. We now provided clear evidence that BRCA1 interacts at the endogenous level also with H1.2 by both coimmunoprecipitation experiments and PLA assay (revised Fig. 1F and S1C).
2) The authors showed that expression of siRNA resistant GFP-H1.3 rescue BRCA1 recruitment. Could they see a similar effect with another histone H1 gene overexpression? this would strengthen their finding that this effect is not H1.3-specific.

To address this reviewer comment we generated U2OS cell lines stably expressing siRNA-resistant GFP-H1.1 and GFP-H1.4 and showed that expression of GFP-H1.1 and GFP-H1.4 (upon silencing of endogenous H1) is able to rescue BRCA1 chromatin recruitment. This data points to a non H1.3-specific mechanism for BRCA1 recruitment to stalled replication forks.
3 4) are there other potential CK2 sites involved which might explain authors results? is this interaction reduced by CK2 inhibition for example? (this should be at least discussed) We appreciate the reviewer's comment. To strengthen our manuscript and address the reviewer comment (as well as reviewer 2 comment) we have now further investigated the mechanistic basis of the H1-BRCA1 interaction. In particular, our new data suggest that interaction between BRCA1 and histone H1 requires both the C-terminal domain (containing the CK2 phosphorylation site S1336) and the ring domain of BRCA1 (revised Fig. S1A). These data suggest that interaction between H1 and BRCA1 involves multiple sites and/or indirect partners (e.g. BARD1). In addition to this, we also show that silencing of RNF8 strongly decrease the HU-dependent interaction between BRCA1 and H1, pointing to an important role for H1 ubiquitination in regulating BRCA1 recruitment to stalled replication forks. We discussed these results in the revised version of our manuscript. : Fig 2b. Contrast should be increased as gH2AX staining is hardly visible.

5) Minor comment
We thank the reviewer for the comment. We have now increased the contrast of γH2AX staining in the revised version of our manuscript. Thank you for submitting your revised manuscript entitled "The linker histone H1-BRCA1 axis is a crucial mediator of replication fork stability". We would be happy to publish your paper in Life Science Alliance pending final revisions necessary to meet our formatting guidelines.
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